Chimeric molecule useful in immunotherapy for leishmaniasis, which includes a fragment of the pfr1 protein of leishmania infantum with specific immunodominant epitopes

ABSTRACT

The present invention claims an isolated nucleotide sequence characterized by encoding the PFR1 protein of  Leishmania infantum  or a fragment thereof. This PFR1 protein or a fragment thereof comprises at least a selected immunodominant epitope between the following group: SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8, where the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response in an animal, against the kinetoplastids causing the leishmaniasis disease. The immunodominant epitopes are cytotoxic T-lymphocyte activators and they present a high binding affinity for A2 type MHC Class I molecule.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/234,075, filed Jun. 12, 2014, which is a 371 application ofInternational Patent Application No. PCT/ES2012/070541, filed Jul. 17,2012, which claims the benefit of Spanish Patent Application No.P201131257, filed Jul. 21, 2011, the disclosures of which are herebyincorporated by reference herein in their entirety.

The present invention is found within the chemical and pharmaceuticalsector, and in the areas of medicine, molecular biology, immunology,parasitology, and veterinary, and refers to an immunological tool forcombating leishmaniasis, a disease caused by different species belongingto the genus Leishmania.

SEQUENCE LISTING

A Sequence Listing comprising SEQ ID NOS:1-13, has been provided incomputer readable form (.txt) as part of this application, and isincorporated by reference herein in its entirety as part of thisapplication.

STATE OF THE ART

The species of the genus Leishmania, Protozoan intracellular parasites,belonging to the order Kinetoplastida Trypanosomatidae family, aredistributed by tropical and subtropical regions of the world, causing abroad spectrum of clinical symptoms, called leishmaniasis. Sorting bythe causative species, leishmaniasis can be classified as cutaneous,mucocutaneous or visceral (in reference to their tissue tropism and theclinical causing symptoms). The incidence of leishmaniasis has increasedenormously in recent years, so that it is now endemic in 88 countriesacross all continents and it is claimed that there are 350 millionpeople at risk of contracting the disease (WHO report,who.int/ctd/html/leisdis.html). It is estimated that worldwide there areabout 12 million people affected by leishmaniasis. Accounting 2 millionnew cases annually, 500,000 are visceral leishmaniasis (VL). Thisdisease is caused by L. infantum and L. donovani and symptoms of thedisease include intermittent fever, anemia, splenomegaly, hepatomegaly,and lymphadenopathy. The outcome of the VL is usually death, caused by aconcomitant infection, given the weakness of the immune system of theaffected patient. Leishmania/HIV co-infection has revealed in recentyears as a major health concern for the Mediterranean area countries. Inthese countries, the main host is the dog, acting as a reservoir of thedisease for humans (Alvar et al. 2004 Adv Parasitol. 57:1-88).

Vaccination is the most efficient medical treatment to prevent mortalityand morbidity due to infectious agents. Despite this, currently thereare very few high-effective vaccines against pathogens against whichthere is an effective vaccine. Front of the different Leishmaniaspecies, in recent years, it has been tested the use of antigen vaccinesor thereof with disparate results (Kedzierski et al. 2010. J Glob InfectDis. 2(2):177-85; Kedzierski et al. 2006. Parasitology, 133:87-112;Requena et al. 2004. Expert Opin Biol Ther. 4(9):1505-17). However, noneof the attempts to obtain a vaccine in humans has been fullysatisfactory so far.

Another interesting option in the fight against the disease istherapeutic or, in its absence, the immunochemotherapy. Pasteurizedadjuvanted complete parasites have been tested (Convit et al. 2003 MedHyg, 97:469-72), as well as a mixture of the parasite antigens (Badaroet al. 2006 J Infect Dis, 194:1151-9), but, as in the case ofvaccination, any ultimate success has failed (El-On J. 2009. Isr MedAssoc J, 11(10):623-8).

As it was outlined before, the dog is the main reservoir of the disease,so that several attempts have also been made from the immunologicalpoint of view in the fight against this human but also veterinary healthproblem. In fact, leishmaniasis is a serious parasitic disease in thedog. The most common clinical symptom is loss of hair, especially aroundthe eyes, ears, and nose. In a more advanced stage of the disease thedog lose weight but not appetite and they are common the presence ofwounds in the skin, especially on the head and legs, as well as symptomsrelated to kidney failure. The disease is endemic in large parts ofSpain, as well as in most of the countries of the Mediterranean region.

Likewise in humans, there have been made numerous attempts to controlthe disease from different angles, both prophylactic and immunotherapy,without a completely satisfactory success so far (Alvar et al. 2004. AdvParasitol. 57:1-88; Reis et al. 2010 Trends Parasitol. 26(7):341-9).

In any case, either human or canine leishmaniasis, one of the mostpromising ways of control of leishmaniasis points to the use of DNAvaccines that carry multiple genes coding for specific leishmaniaantigens or chimeric recombinant proteins containing different antigensof the parasite (De Oliveira et al. 2009 Parasitol Int. 58(4):319-24;Palatnik-de-Sousa, 2008. Vaccine 26:1709-1724).

PFR proteins (Paraflagellar Rod proteins) represent a family of relevantspecific antigens of tripanosomatids that are located in theparaflagellar region of these parasites (Fouts et al., 1998 J Biol Chem,273(34):21846-21855; Clark et al., 2005. Parasitol Res. 96(5):312-320).Some members of this family of antigens, PFR1-3 proteins, stand out fortheir high immunogenicity (Michailowsky et al., Infect Immun71(6):3165-3171). Immunization of mice with PFR1 and PFR2 proteinspurified from T. cruzi induces a Th1 immune response capable ofreducing, in experimental infection tests, T. cruzi load in the hearttissue of mice immunized and infected with the T. cruzi parasite (Morellet al. 2006 Vaccine, 24:7046-7055). These results show that PFR proteinsmay be suitable candidates for use as vaccines. According to theseresults, it has been found that lymphocytes CD4+ isolated from miceimmunized with PFR are capable of activating parasite-infectedmacrophages causing the death of the parasite by NO releasing(Wrightsman et al. 2000 Vaccine 18(14):1419-27, Miller et al. 1997 JImmunol 158(11):5330-7), and also being able to reduce the level ofparasites in blood, in the absence of B cells, but not in the absence offunctional CD4+ and CD8+ T lymphocytes as well. From the point of viewof gene immunization, different paraflagellar proteins of differentkinetoplastids have been studied. The immunogenic and protectivecapacity of L. mexicana PFR2 protein after their inoculation as both DNAand recombinant protein has been demonstrated (Saravia et al. 2005Vaccine 23:984-995). Immunization of mice with DNA vectors containingthe gene that encodes the PFR2 protein of T. cruzi alone or fused to theHsp70 protein of the same pathogen induces high levels of IgG2a typeanti-PFR antibodies. However, only the immunization with the chimericvaccine stimulates the production of IL-12 and IFN-γ, and causes thedecrease of IL-4 producing cells, triggering a protective responseagainst T. cruzi (Morell et al. 2006 Vaccine 24:7046-7055).

Hsp70 protein belongs to the heat shock proteins (HSP) family, which arehighly conserved among the different species (eukaryotes andprokaryotes). They are fundamental in maintaining cellular homeostasisby their role as chaperone (Smith, Whitesell et al. 1998 PharmacologicalReviews 50(4):493-513). They are very interesting for their ability toactivate the immune system, highlighting the Hsp70 family by itsimmunological versatility. Hsp70 proteins obtained from tumor cells orvirus-infected cells are capable of activating CD8+ CTL response both invivo and in vitro against various expressed antigens in the cells ofwhich the immunogenic protein has been purified from (Srivastava. 2002Nat Rev Immunol 2:185-194; Wu et al., 2005. Cancer Res.65(11):4947-4954). Thus, the extracellular Hsp70 can form complexes withantigenic peptides and activate APCs at the same time. This interactiontriggers a cascade of events, gathering processes of peptidescross-presentation to CD8+ restricted MHC I and T CD4+ cells restrictedMHC II, secretion of proinflammatory cytokines, and functional andphenotypic maturation of dendritic cells (DCs) (Asea et al. 2000 Nat Med6:435-442; Basu et al. 2001 Immunity 14:303-313; Harmala et al. 2002 JImmunol 169:5622-5629; Tobian et al. 2004 J Immunol 173:5130-5137).

On in vitro tests carried out in our laboratory, the Trypanosoma cruziHsp70 protein has shown to have a unique stimulator effect on spleen andganglion cells of naive mice (Marañón et al., 2000. Int. Immunol.12(12):1685-1693), resulting in a quick and intense stimulation of Tcells. In addition, this protein is able to induce in vivo and in vitro,against the associated hapten, a mixed immune response (IgG1 and IgG2a)which, interestingly, turns out to be independent of TLR2 and TLR4receptors (Qazi et al. 2007 Vaccine 25(6):1096-1103). This T. cruziHSP70 protein has also proven to be capable of triggering a specificresponse against KMP11 protein in mice when they are immunized with avector bearing the sequences that encode for both proteins, byactivating the production of IgG2a type specific antibodies againstKMP11 (Thomas, Olivares et al. 2000 DNA and Cell Biology 19(1):47-57;Thomas, Olivares et al. 2000 Acta Tropica 75(2):203-210). Moreover, miceimmunized with the KMP11-HSP70 fusion protein, but not those immunizedwith the isolated KMP11 protein, induce a cytotoxic lymphocyte responseagainst Jurkat-A2/Kb cells expressing the KMP11 protein as well asagainst those cells loaded with KMP11-derived peptides of provenaffinity to A2 molecule. Similar achievements have been obtained usingthe Leishmania genus itself, administering Hsp70 together to the gp63metalloproteinase of Leishmania donovani (Kaur et al. 2011 ParasiteImmunol. 33(2):95-103).

To fight against different species of Leishmania, in recent years,vaccines comprising the inactivated whole parasite and/or modifiedantigens or fragments thereof, as purified as recombinant, have beentested with different results. Thus, according to our own data, none ofthe previous attempts to obtain a fully successful vaccine for humans ordogs has been achieved so far. Even though chemotherapy currently in usehas shown some effectiveness in many cases, it is unable to generate anenough parasite clearance, necessary for a full control and eradicationof the disease.

Five years ago, it was carried out the identification of A*0201molecule-restricted T cell binding epitopes contained in the PFR1protein, as well as the generation of chimeric molecules (DNA vaccines)formed by the sequences coding for the L. infantum PFR1 protein orfragments derived from this protein, for their use both isolated andassociated to the HSP70 protein and/or fragments thereof, as a carriermolecule. In addition, it was studied in immunized mice thePFR1-specific immune response induced by the referred chimericmolecules.

There are previous studies in the literature not recommending the use ofpeptides isolated from the PFR proteins of T. cruzi for induction of CTLresponse. In particular, Wrightsman R. A. et al., published an articlein 2002 where various peptides isolated from T. cruzi PFR proteins weretested and evaluated whether these peptides were efficiently processedand presented, within the context of a MHC-Class I, in the course of anexperimental infection with this parasite and whether they wererecognized by cytotoxic T cells (CTLs) in immunized animals (Wrightsmanet al. 2002 Parasite Immunol. 2 (8):401-12). In this study, PFR1epitopes capable of binding to murine HLA class I molecules (H-2 Kb andH-2Db) are identified, but the authors conclude that mice immunizationwith PFR1 or PFR3 proteins does not induce CTLs against these proteinsnor against the identified peptides.

Therefore, in view of the state of the art information, currently thereis a need to demonstrate the efficiency of chimeric molecules (DNAand/or recombinant vaccines), formed by the coding sequence of antigenicproteins of L. infantum PFR1. The PFR1 protein comprises specificepitopes capable of inducing a CTL response, both isolated andassociated with the HSP70 protein and/or fragments thereof, as carriermolecule. At the same time, it is necessary to test its efficacy andsafety as prophylactic and therapeutic vaccine against Leishmaniainfection, both in humans and dogs.

BRIEF DESCRIPTION OF THE INVENTION

The present invention represents a useful tool in the fight againstanimal and human leishmaniasis. It is based on the use of the PFR1protein-coding nucleotide sequences of Leishmania infantum or fragmentsthereof containing a series of peptides, alone or in conjunction withthe Hsp70 protein of Trypanosoma cruzi, as well as plasmids containingtheir genetic encoding sequences. These peptides contained within thePFR1 protein of L. infantum correspond to immunodominant epitopesrecognized specifically by CD8+T lymphocytes showing cytotoxic capacity,induced by the aforementioned protein used as immunogenicity agent.Where a preferential, the identified epitopes are presented by the humanmolecule HLA-A*0201. By means of these genetic and protein molecules thepresent invention provides a cellular and humoral immune response in thehost against the protozoan parasite, with capacity for control,significantly, Leishmania infection or prevent it. The molecules objectof the present invention are relevant for their role in immunotherapyagainst leishmaniasis used both as a preventive vaccine as therapeutic(isolated or in combination with various chemotherapy). Thus, the use ofthese molecules represents an industrial advantage over the lack ofreally effective preventive vaccines against this parasitic diseasealready in the market along with the difficulties and inconveniencesthat presents more traditional pharmacological treatments. Although theyshow some effectiveness in the majority of cases, in many other(especially dogs) do not lead to a complete clearance of the parasite,the whole treatments are quite expensive, they often have many sideeffects, and there is an increasing occurrence of cases of resistance,among their main setbacks.

DETAILED DESCRIPTION OF THE INVENTION

The present invention claims an isolated nucleotide sequencecharacterized by encoding the PFR1 protein of Leishmania infantum or afragment thereof. This PFR1 protein or a fragment thereof comprises atleast a selected immunodominant epitope between the following group: SEQID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ IDNo: 6, SEQ ID No: 7 and SEQ ID No: 8, where the immunodominant epitopeis able to induce an antigen-specific T cell cytotoxic immune responsein an animal, against the kinetoplastids causing the leishmaniasisdisease. The immunodominant epitopes are cytotoxic T-lymphocyteactivators and they present a high binding affinity for A2 type MHCClass I molecule.

Within the context of the present invention “immunodominant epitope” isconsidered a fragment peptide capable of being recognized by lymphocytereceptors and of generating an epitope-specific cellular immuneresponse.

In a preferred embodiment of the present invention the expression“animal” refers to a human or a pet, and more preferably a dog.

In a preferred embodiment of the present invention “nucleotide sequence”refers to any nucleotide sequence containing DNA, preferably genomicDNA, synthetic DNA, RNA, in vitro transcribed RNA, messenger RNA, beingsense or antisense sequences. Therefore, the present invention refers tothese sequences regardless of their obtaining conditions, thus includingsequences obtained from a biological sample by cloning or by chemicalsynthesis or by enzymatic processes.

In a preferred embodiment of the present invention “nucleotide sequenceencoding for a protein or a fragment thereof” refers to a nucleotidesequence that is capable, under adequate control of expression by theircorresponding regulation elements (promoters, enhancers, transcriptionsites, etc.), of transcribing and translating one amino acid sequence ofthe protein of interest or a fragment thereof. The protein of interestas well as the fragments thereof referred to in the present invention ischaracterized by their amino acids sequence, but they can also becharacterized by the cell where they are expressed, their maturationprocess and the existing environmental conditions during expression.

In a preferred embodiment of the present invention the term “amino acidsequence” refers to a sequence of an oligopeptide, peptide, protein, ortheir fragments, which are naturally or synthetically produced. As asequence encoded by a nucleotide sequence is referred within the presentinvention, the term is not limited to the complete amino acid sequences,or native amino acid sequences associated with the aforementionedprotein molecule, it is also referring to the variations that may suffersuch native amino acid sequence.

In a particular embodiment, the present invention claims the nucleotidesequence defined above, encoding for the 1-595 amino acids of Leishmaniainfantum PFR1 protein, determined by the SEQ ID No: 12. This fragmentencodes the complete protein.

In another particular embodiment, the present invention claims thenucleotide sequence defined above, encoding for the 160-595 amino acidsof Leishmania infantum PFR1 protein, determined by the SEQ ID No: 9.

In another particular embodiment, the present invention claims thenucleotide sequence defined above, encoding for the 160-548 amino acidsof Leishmania infantum PFR1 protein, determined by the SEQ ID No: 10.

In another particular embodiment, the present invention claims thenucleotide sequence defined above, encoding for the 160-385 amino acidsof Leishmania infantum PFR1 protein, determined by the SEQ ID No: 11.

The present invention also protects a chimeric molecule comprising:

-   (a) a nucleotide sequence defined above, that is preferably fused to-   (b) a nucleotide sequence encoding for the Hsp70 protein of    Trypanosoma cruzi, or fragments thereof;    wherein the chimeric molecule is able to induce an antigen-specific    cytotoxic T cell immune response in an animal, against the    kinetoplastids causing the leishmaniasis disease.

In a preferred embodiment of the present invention “chimeric molecule”refers to a DNA molecule containing nucleotide sequences from twodifferent species, preferably those species are Leishmania infantum andTrypanosoma cruzi.

In a particular embodiment, the present invention claims the chimericmolecule defined above, carrying the nucleotide sequence encoding forthe Hsp70 protein of Trypanosoma cruzi, determined by SEQ ID No: 13, orfragments of the HSP70 with carrier activity.

In another particular embodiment, the present invention claims thechimeric molecule defined above, incorporating at the 3′ end of the geneencoding fragment for the PFR1 protein fragment corresponding from 160to 595 amino acids, characterized by the SEQ ID No: 9, and a nucleotidesequence encoding for the Hsp70 protein of Trypanosoma cruzi, determinedby the SEQ ID no.: 13 or HSP70 fragments with carrier activity.

In another particular embodiment, the present invention claims thechimeric molecule defined above, incorporating at the 3′ end of the geneencoding fragment for the PFR1 protein fragment corresponding from 160to 548 amino acids, characterized by the SEQ ID No: 10, and a nucleotidesequence encoding for the Hsp70 protein of Trypanosoma cruzi, determinedby the SEQ ID no.: 13 or HSP70 fragments with carrier activity.

In another particular embodiment, the present invention claims thechimeric molecule defined above, incorporating at the 3′ end of the geneencoding fragment for the PFR1 protein fragment corresponding from 160to 365 amino acids, characterized by the SEQ ID No: 11, and a nucleotidesequence encoding for the Hsp70 protein of Trypanosoma cruzi, determinedby the SEQ ID no.: 13 or HSP70 fragments with carrier activity.

In another particular embodiment, the present invention claims thechimeric molecule defined above, incorporating at the 3′ end of the geneencoding fragment a nucleotide sequence encoding for the Hsp70 proteinof Trypanosoma cruzi, determined by the SEQ ID no.: 13 or HSP70fragments with carrier activity.

The present invention also claims a recombinant or expression vectorcomprising:

-   (a) a nucleotide sequence defined above, or-   (b) a chimeric molecule defined above.

The present invention also claims a recombinant plasmid comprising thevector defined above.

Another embodiment of the present invention claims a composition,preferably pharmaceutical or immunogenic, comprising:

-   (a) a nucleotide sequence defined above, or-   (b) a chimeric molecule defined above.

Another embodiment claimed by the present invention concerns the use ofthe pharmaceutical or immunogenic composition defined above, for thetreatment and/or prevention of kinetoplastids infection that causesleishmaniasis disease in an animal.

Another embodiment claimed by the present invention concerns a methodfor the manufacture of a preventive or therapeutic vaccines for thetreatment and/or prevention of kinetoplastids infection causing theleishmaniasis disease, through the use of the pharmaceutical orimmunogenic composition defined above. Such use or method ischaracterized by comprising the following stages:

-   a) identifying the encoding sequences of interest, preferably    identify at least one nucleotide sequence defined above,-   b) amplifying at least one nucleotide sequence identified in a) by    PCR using genomic DNA from the kinetoplastid that causes the    leishmaniasis disease, and oligonucleotides containing restriction    enzymes sites that allow the amplicon direct cloning into    prokaryotic and eukaryotic expression vectors after its digestion    with these restriction enzymes,-   c) cloning at least one nucleotide sequence amplified in b) for the    production of the protein encoded by the said nucleotide sequence,-   d) purifying at least one of the proteins obtained in c), also known    as recombinant proteins (chimeras or not), by affinity    chromatography, and-   e) producing at least one endotoxin-free DNA vector for a safe    inoculation of a preventive or therapeutic vaccine for the treatment    and/or prevention of kinetoplastids infection causing the    leishmaniasis disease.

Another embodiment claimed by the present invention concerns the use ofa nucleotide sequence, a chimeric molecule, a recombinant vector, arecombinant plasmid, or a pharmaceutical or immunogenic composition, asdefined above, as markers in methods for controlling the degree ofinfection of kinetoplastids that cause leishmaniasis disease in ananimal.

Preferably, the present invention claims the use of a nucleotidesequence, a chimeric molecule, a recombinant vector, a recombinantplasmid, or a pharmaceutical or immunogenic composition, defined above,to generate a protective immunological memory against the infection ofthe kinetoplastids causing the leishmaniasis disease in an uninfectedanimal.

More preferably, the present invention claims the use of a nudeotidesequence, a chimeric molecule, a recombinant vector, a recombinantplasmid, or a pharmaceutical or immunogenic composition, defined above,to clarify or generate partial or total clearance of the kinetoplastidscausing the leishmaniasis disease of the tissues in an infected animal.

The present invention has mainly two clear options of immuno-therapeuticapplications, a preventive vaccination and its use in immune therapiesagainst infection by Leishmania. Likewise, the molecules/products objectof the present invention can be used in the form of recombinantproteins, as DNA plasmids (genetic vaccine), or in a combined form.

Thus, a first implementation of the claimed products would be the use ofthese molecules to generate protective immunological memory againstinfection by the parasite protozoan mentioned, either in humans or dogs,getting control of the disease in endemic areas (vaccination). Anotherapplication would be the use of these molecules as immunotherapy inindividuals already infected, in order to enhance the immune response ofthe host against the parasite and control it. This treatment can beisolated or in combination with other existing chemotherapeutic, chasingthe total clearance of the parasite. Thus, activating and modulating theimmune response of the host against the parasite and especially byinducing a T cytotoxic antigen-specific response the immune system coulderadicate the parasites stationed in tissues, such as bone marrow orspleen that the majority of chemotherapeutic treatments fail to clarify.

Throughout the description and claims the word “comprise” and itsvariants do not exclude other technical features, additives, componentsor steps. For experts in the field, other objects, advantages andfeatures of the invention come off as part of the description and aspart of the practice of the invention. The following figures andexamples are provided for illustration and they are not intended tolimit the scope of the present invention.

FIGURE LEGENDS

FIG. 1. Purified PFR-1 recombinant protein was electrophoresed in a 10%SDS-PAGE gel and visualized by coomasie blue staining. MW. Proteinmolecular weight marker.

FIG. 2. Measurement of Nitric Oxide (NO) concentration in thesupernatant of macrophages culture stimulated with lipopolysaccharide(LPS); lipopolysaccharide+polymyxin B (LPS+PolB); purified PFR-1 protein(PFR-1); purified PFR-1 protein+polymyxin B (PFR1+PolB); L-NG-monomethylarginine citrate (LNMMA); purified PFR-1 protein+L-NG-monomethylarginine citrate (PFR1+LNMMA). Concentration is expressed in pmol/litre.

FIGS. 3A to 3C. Western blot analysis of PFR-1 and PFR1-Hsp70 proteinexpression in COS-7 cells transfected with the pCMV4 empty vector (lane2) and pCMV4 PFR1 (lane 3); pCMV4 PFR1-Hsp70 (lane 4) and pCMV4PFR1-Th70 (lane 5) constructs. No transfected cells are used as control(lane 1). Proteinmolecular weight marker (Lane 6). Poly clonal antibodyagainst LiPFR1 (FIG. 3A), TcHsp70 (FIGS. 3B and 3C).

FIGS. 4A to 4B. FIG. 4A. IgG antibody level against PFR-1 recombinantprotein in sera from mice inoculated with saline solution and immunizedwith pCMV4 PFR-1 and pCMV4 PFR1Hsp70 vectors, 14 (grey Bars) and 40 days(black bars) after the fourth immunization. FIG. 4B. Level of IgG1 (greybars) and IgG2a (black bars) antibodies specific of the PFR-1recombinant protein detected in the immunized mice referred in FIG. 4Aat 14 and 40 days post fourth immunization. Bars represent the meanvalue of optical density of each group.

FIGS. 5A to 5D. Antibody level against Leishmania soluble antigens (SLA,FIGS. 5A, 5B and 5C) and PFR1 recombinant protein (FIG. 5D) in sera fromC57BL/6 mice inoculated with saline solution (SS) and, pCMV4 plasmid(pCMV4) or immunized with pCMV4 PFR-1 (PFR1), pCMV4 PFR-1-Hsp70 (70c)and pCMV4 PFR-1 truncated Hsp70 (70t) recombinant vectors, 14 and 21days after challenge with Leishmania infantum infective promastigotes.Bars represent the mean of optical density of each group.

FIG. 6. Lymphoproliferative response to PFR-1 protein in mice inoculatedwith saline solution (SS) and pCMV4 empty vector (pCMV4) and immunizedwith pCMV4-PFR1 (PFR1), pCMV4 PFR1-HSP70 (70c) and pCMV4 PFR1-TruncatedHsp70 (70t). Stimulation index was calculated as [(arithmetic mean ofcpm (stimulated culture)—arithmetic mean of cpm (controlculture))/arithmetic mean of cpm]. The results represent the mean andstandard deviation of three independent immunization experiments.

FIG. 7. Nitric Oxide production (NO) by peritoneal macrophages fromimmunized C57BL/6 mice inoculated with saline solution (SS) and pCMV4empty vector (pCMV4) or immunized with pCMV4 PFR1 (PFR1), pCMV4PFR1-Hsp70 (70c) and pCMV4 PFR1-truncated Hsp70 (70t) constructs afterLeishmania infantum challenge in response to lipopolysaccharide (LPS),culture media (Medium), Leishmania soluble antigens (SLA) and PFR1recombinant protein.

FIG. 8. Parasite burden in spleen, liver and bone marrow from miceinoculated with saline solution (SS) and empty vector pCMV4 (pCMV4) orimmunized with pCMV4 PFR1 (PFR1), pCMV4 PFR1-Hsp70 (70c) and pCMV4PFR1-truncated Hsp70 (70t) recombinant vectors at 14 and 21 days postchallenged with Leishmania infantum infective promastigotes.

FIG. 9. Binding assay of HLA*02:01-restricted PFR1-derived peptides toTAP-deficient T2 cells. Percentage of maximal complex stabilization wascalculated with the HB-ENV₃₃₄₋₃₄₂ peptide fluorescence index as areference. The binding of each peptide was determined at differentconcentrations of each peptide.

FIG. 10. Cytotoxic activity of the CD8⁺ T lymphocytes specific for theeight selected PFR1 peptides evaluated by the secretion of GzB throughELISPOT in splenocytes from B6-A2/K^(b) mice inoculated with salinesolution (SS) or immunized with pCMV4 PFR1 (PFR1) or pCMV4 PFR1-Hsp70(PFR1-HSP70) recombinant vectors. Spots were visualized using a KSELISPOT device (Zeiss). Only large spots with fuzzy borders were scoredas spot-forming cells (SFC). Responses were considered significant if(i) a minimum of 150 SFC/106 splenocytes were detected after subtractionof the negative control (splenocytes without peptide), and additionally,(ii) the response was at least over two fold the negative control.

FIG. 11. Cytotoxic activity of the CD8⁺ T lymphocytes specific for theeight selected PFR1 peptides evaluated by the secretion of GzB throughELISPOT in splenocytes from B6-A2/Kb mice infected with Leishmaniainfantum infective promastigotes. Non-inoculated animals were used ascontrol. Spots were visualized using a KS ELISPOT device (Zeiss). Onlylarge spots with fuzzy borders were scored as spot-forming cells (SFC).Responses were considered significant if (i) a minimum of 250 SFC/106splenocytes were detected after subtraction of the negative control(splenocytes without peptide), and additionally, (ii) the response wasat least over two fold the negative control.

FIG. 12. Cytotoxic activity of the CD8+T lymphocytes specific for theeight selected PFR1 peptides evaluated by the secretion of GzB throughELISPOT in hepatocytes from B6-A2/Kb mice infected with Leishmaniainfantum infective promastigotes. Non-inoculated animals were used ascontrol. Spots were visualized using a KS ELISPOT device (Zeiss). Onlylarge spots with fuzzy borders were scored as spot-forming cells (SFC).Responses were considered significant if (i) a minimum of 75 SFC/106hepatocytes detected after subtraction of the negative control(hepatocytes without peptide), and additionally, (ii) the response wasat least over two fold the negative control.

FIG. 13. The prediction of potential HLA-A*02:01 ligands contained in L.Infantum PFR1 protein was carried out through the screening of thededuced amino acid sequence of PFR1 gene using three HLA-A2-bindingaffinity algorithms: SYPFEITHI (www.syfpeithi.de), RANKPEP(immunax.dfci.harvard.edu/Tools/rankpep.html) and BIMAS (theoreticalhalf-time dissociation, www.bimas.cit.nih.gov/molbio/hla_bind/).

FIGS. 14A to 14B. FIG. 14A. IgG antibody level against PFR1 recombinantprotein in sera from mice inoculated with saline solution and immunizedwith pCMV4 436aaPFR1 and pCMV4 436aaPFR1Hsp70 vectors, 14 (black Bars)and 40 days (grey bars) after the fourth immunization. FIG. 14B. Levelof IgG1 (black bars) and IgG2a (grey bars) antibodies specific of thePFR-1 recombinant protein detected in the immunized mice referred inFIG. 14A at 14 and 40 days post fourth immunization. Bars represent themean value of optical density of each group.

FIG. 15. Lymphoproliferative response to PFR-1 protein in miceinoculated with saline solution (SS) or immunized with pCMV4 436aaPFR1(436aaPFR1) and pCMV4 436aa PFR1-HSP70 (436aa PFR1 HSP70). Stimulationindex was calculated as [(arithmetic mean of cpm (stimulatedculture)—arithmetic mean of cpm (control culture))/arithmetic mean ofcpm]. The results represent the mean and standard deviation of threeindependent immunization experiments.

FIG. 16. Cytotoxic activity of the CD8+ T lymphocytes specific for theeight selected PFR1 peptides evaluated by the secretion of GzB throughELISPOT in splenocytes from B6-A2/Kb mice inoculated with salinesolution or immunized with pCMV4 436aaPFR1 or pCMV4 436aa PFR1-Hsp70recombinant vectors. Spots were visualized using a KS ELISPOT device(Zeiss). Only large spots with fuzzy borders were scored as spot-formingcells (SFC). Responses were considered significant if (i) a minimum of150 SFC/106 splenocytes were detected after subtraction of the negativecontrol (splenocytes without peptide), and additionally, (ii) theresponse was at least over two fold the negative control.

BIBLIOGRAPHY

-   1—Alvar et al., 2004. Adv Parasitol. 57: 1-88-   2—Kedzierski et al., 2010 J Glob Infect Dis. 2 (2): 177-85-   3—Kedzierski et al., 2006 133: 87-112, Parasitology-   4—Requena et al., 2004 Expert Opin Biol Ther. 4 (9): 1505-17-   5—Convit et al., 2003. Med Hyg, 97: 469-72-   6—Badaro et al., 2006 J Infect Dis, 194: 1151-9-   7—On j. 2009. ISR Med Assoc J, 11 (10): 623-8-   8—Alvar et al., 2004. Adv Parasitol. 57: 1-88-   9—Reis et al., 2010. Trends Parasitol. 26 (7): 341-9-   10—de Oliveira et al., 2009 Parasitol Int. 58 (4): 319-24-   11—Palatnik-de-Sousa, 2008. Vaccine 26: 1709-1724-   12—Fouts et al., 1998 J Biol Chem, 273 (34): 21846-21855-   13—Clark et al., 2005. Parasitol Res. 96 (5): 312-320-   14—Michailowsky et al., Infect Immun 71 (6): 3165-3171-   15—Morell et al., 2006 Vaccine, 24: 7046-7055-   16—Wrightsman et al., the. 2000 Vaccine 18 (14): 1419-27-   17—Miller et al., 1997 J Immunol 158 (11): 5330-7-   18—Saravia et al., 2005 Vaccine 23: 984-995-   19—Morell et al., 2006 Vaccine 24: 7046-7055-   20—Smith, Whitesell et al., 1998 Pharmacological Reviews 50 (4):    493-513-   21—Srivastava, 2002 Nat Rev Immunol 2: 185-194-   22—Wu et al., 2005. Cancer Res. 65 (11): 4947-4954-   23—Asea et al., 2000 Nat Med 6: 435-442-   24—Basu et al., 2001 Immunity 14: 303-313-   25—Harmala et al., 2002 J Immunol 169: 5622-5629-   26—Tobian et al., 2004 J Immunol 173: 5130-5137-   27—Marañón et al., 2000. Int. Immunol. 12 (12): 1685-1693-   28—Qazi et al., 2007 Vaccine 25 (6): 1096-1103-   29—Thomas, Olivares et al., 2000 DNA and Cell Biology 19 (1): 47-57-   30—Thomas, Olivares et al., 2000 Acta Tropica 75 (2): 203-210-   31—Kaur et al., 2011 Parasite Immunol. 33 (2): 95-103-   32—Wrightsman R. A. et al., 2002 Parasite Immunol. 24 (8): 401-12-   33—Buffet et al., 1995, Antimicrob. Agents Chemother. 39 (9):    2167-2168

EXAMPLES

The following specific examples that are provided in this patentdocument are intended to illustrate the nature of the present invention.These examples are only for illustrative purposes and should not beinterpreted as limitations to the invention that is claimed here.Therefore, the examples described below illustrate the invention withoutlimiting the field of application of the same.

Example 1

1.1. The PFR1 Protein Induces the Expression of Nitric Oxide (NO).

PFR1 protein of Leishmania infantum is encompassed within the family ofcharacteristic proteins of paraflagellar rod proteins fromkinetoplastids. In FIG. 1, we show the PFR1 recombinant protein of L.infantum expressed in a prokaryotic expression system and purified byaffinity chromatography. For this protein we have demonstrated that ithas the ability to activate the production of nitric oxide (NO), one ofthe main mechanisms that the macrophages have to eliminate pathogensthat have phagocytosed into alveolar macrophages of naïve rat, i.e.without any treatment or previous infection. This activation of nitricoxide production confers to the aforementioned protein a relevantimmunological feature because NO favors the clearance of the amastigotesof Leishmania that multiply within the host macrophages. In FIG. 2, thevalues of NO production by these alveolar macrophages are collected bystimulating them with LPS (control) and the PFR1 protein. Interestingly,unlike the one detected for PFR1 protein, the activation observed forLPS is inhibited by the presence of polymyxin B (LPS activity-inducedinhibitor), which discards any contamination with LPS from the PFR1recombinant protein. On the other hand, the addition of L-N⁶-monomethylArginine (LNMMA), an inhibitor of the enzyme nitric oxide synthase,inhibits the NO activation induced by the PFR1 protein, indicating themechanism of action of this protein. Similar results are obtained by thefusion PFR1-HSP70 protein.

1.2. Expression of PFR1 Protein in Eukaryotic Cells.

The determination of the correct expression of the proteins to study ineukaryotic cells is determined by the analysis of COS-7 transfectedcells with genes encoding for PFR1 protein and chimeric fusion proteinsHSP70-PFR1 and PFR1-H70T cloned into pCMV4 plasmid. The visualization ofdifferent proteins is made by Western blots analysis of cells extractstransfected with the respective abovementioned plasmids and inducedPFR1-protein polyclonal antibodies. Thus, in FIG. 3 is observed,respectively, the presence of recognition bands with sizes of 70 kDa(corresponding to the PFR1 protein in the cells lane with the pCMV4PFR1), 140 kDa in the lane of COS-7 cells transfected with the pCMV4PFR1-Hsp70 plasmid and 96 kDa in the cells lane with the pCMV4PFR1-Hsp70T plasmid. Cells containing the empty pCMV4 plasmid are notdetected.

1.3 Antigen-Specific Humoral Response Induced by the Tested Molecules.

As example we show the results obtained after intramuscularlyimmunization in groups of 12 mice of the C57BL/6 strain, with 100 μg ofdifferent plasmids under study, as well as negative controls (emptyplasmid and saline solution). Each mouse was immunized 4 times every twoweeks. Six weeks after the fourth immunization, six mice in each groupwere challenged intravenously with 105 infective promastigotes of theJPCM5 (MCAN/s/98/LLM-724) strain of L. infantum.

To analyze the humoral response generated in different groups of mice,blood samples were collected two weeks after each immunization andspecific antibody measured. The obtained results indicate the presenceof anti-PFR1 IgG antibodies 15 days post-second immunization in thegroup of mice immunized by the isolated PFR1 gene and two weeks afterthe third immunization in those immunized by the fused HSP70-PFR1 orPFR1-HSP70T genes. FIG. 4 shows the results for each molecule at 14 and−40 days post-fourth immunization. IgG levels registered in sera frommice immunized by the PFR1 gene fused to the HSP70 gene were higher thanthose detected in mice immunized with the isolated PFR1 gene, peakingwithin two weeks post-fourth immunization. In all cases the level ofanti-PFR1 antibodies slightly descends after six weeks post-fourthimmunization. The isotype analysis reflects that generated antibodiesshow a clear polarization of the response towards the IgG2a isotype(Th1-type immune response). Similar results were obtained for thePPFR1-H70T molecule.

The results obtained in PFR1 protein humoral response assays and totalantigens of Leishmania (SLA) occurring in mice immunized by themolecules under study and challenged with L. infantum are shown in FIG.5. Analysis of these results indicates that infection with Leishmaniadoes not induce significant variation in IgG levels against PFR1 and thesame polarization towards the IgG2a isotype is observed prior to theinfection.

1.4 Antigen-Specific Cell Response Induced by the Molecules Under Study.

Lymphoproliferation tests were carried out three weeks after the fourthimmunization. The spleens were extracted in sterile conditions and theobtained splenocytes were cultivated in vitro, with an increasingconcentration of the recombinant PFR1 protein (0.4, 2 and 10 μg/ml). Inaddition, in this assay a mitogen (ConA) and unstimulated splenocyteswere included as a positive and negative control, respectively. From theresults obtained, shown in FIG. 6, a significant cellular proliferationrate is observed as the recombinant PFR1 is present and, in addition,this stimulation is dose-dependent in the splenocytes from miceimmunized by the recombinant testing molecules. Interestingly, thisindex was significantly higher in the groups receiving the PFR1 genefused to the HSP70 and HSP70T. The proliferation rate (IE) for thesplenocytes from mice immunized with these fusion molecules areapproximately 25, while the measured index for splenocytes from miceimmunized with the plasmid containing the isolated PFR1 gene is about20. In both cases, above the controls; mice inoculated with the emptyplasmid, IE=13 and saline solution, IE=4. These proliferation rates ofsplenic cells of mice immunized with the testing molecules remain withsimilar values after eight weeks post-fourth immunization. In addition,stimulation capacity is maintained after the challenge, noting a maximumof proliferation rate (cellular response) in mice immunized by theisolated PFR1 gene and after stimulating with 2 μg/ml of the PFR1recombinant protein.

1.5 Expression of Nitric Oxide (NO) in Mice Immunized and Infected by L.infantum.

As shown in FIG. 7, it can be seen a significant greater ability toproduce nitric oxide (NO) in peritoneal macrophages of mice immunizedwith the testing molecules, especially with the fused ones (PFR1-HSP70or PFR1-HSP70T) comparing to the control mice, both stimulated andunstimulated. In addition, this NO releasing ability of the mentionedimmunized groups of mice increases significantly after stimulation withthe recombinant PFR1 protein. On the other hand, such production wassignificantly higher in the infected mice versus the unchallenged ones.

1.6 Determination of the Capability of Inducing Protection Against L.infantum Infection.

The parasite load was analyzed by limiting dilution (Buffet et al.,1995, Antimicrob. Agents Chemother. 39 (9): 2167-2168), in the liver,spleen and bone marrow (target tissues of the parasite), after 14 and 28days post-infection, showing a summary of the results in FIG. 8. Thus,after 14 days post-infection all groups of mice had parasites in theliver, however, in control groups (ss and pCMV4) the parasite load wassignificantly higher than the detected in the groups of animalsimmunized with different testing molecules, showing higher values in atleast one order of magnitude. In addition, unlike the control groups,vaccinated mice with the testing molecules do not show an increase inhepatic parasitic load at 28 days post-infection. In fact, miceimmunized with PFR1 gene fused to the full HSP70 gene (pPFR1-HSP70),showed and important parasite clearance in the liver. The analysis ofthe parasite load in spleen shows a similar pattern. Thus, after 14 dayspost-infection it is observed a significant higher parasite load (atleast one order of magnitude) in splenic tissue of control mice (ss andpCMV4) versus vaccinated mice. In fact, mice vaccinated with PFR1 genefused to the HSP70T gene (pPFR1-H70T) show no parasites in spleen aftertwo weeks post-infection. Regarding bone marrow, only control groups (ssand pCMV4) account parasites, detected just after 28 dayspost-infection. Interestingly, none of the vaccinated mice presentsparasites in this tissue. In summary, all vaccinated groups showed inall analyzed tissues during the infection a significantly lowerparasitic load than the control groups (decrease between two to fourorders of magnitude), pointing that these molecules confer a high levelof protection against L. infantum infection, as intravenouslyadministered.

The analysis of the expression pattern of cytokines of splenocytes ofvaccinated mice versus control groups (cytometry measures in thesupernatant of the cell culture using the Mouse Th1/Th2 Cytokine CBA-BDBiosciences kit) shows the existence of higher levels of TNF-α and IFN-γand with statistical significance in vaccinated mice with PFR1-HSP70 andPFR1-H70T chimeric constructs, stimulated or unstimulated with rPFR1and/or SLA, versus the control groups (P<0.01). However, there is not astatistically significant variation among groups regarding IL-2 or IL-4levels. Likewise, the analysis, after the challenge with Leishmania, ofthe level of the splenic macrophage activation of all the mice groups,problem and control, is measured by the expression pattern of CD80, CD86and CD40 surface molecules. The results show that after 21 dayspost-infection the expression of CD86 and CD40 was significantly higherin the vaccinated group with chimeric constructions PFR1-HSP70 andPFR1-H70T versus the control (P<0.01).

1.7 Identification of T CD8+ Epitopes T in the PFR1 Protein of L.infantum.

The identification of epitopes in the sequence of the PFR1 protein of L.infantum able to be recognized by CD8+ T cells and, as a result ofactivating a cytotoxic antigen-specific response in the host, wascarried out trying to identify epitopes capable of binding to MHC-dass IHLA. To do this, it was selected, by in silico analyses, differentpeptides capable of binding to HLA-A*0201 (expressed in the half of thehuman population), using three programs: SYFPEITHI, RANKPEP, and BIMAS.The deduced sequence of the PFR1 protein of Leishmania infantum (genenumber AY702344) was introduced in each of the programs and it wasselected the algorithm of binding to HLA-A*0201. The first two programsdeliver a score to each peptide based on its theoretical affinity withthe HLA molecule. On the other hand BIMAS scores the stability ofbinding with the HLA molecule, focusing on the theoretical binging time.Combining the results, eight theoretical epitopes of high bindingaffinity to class I HLA molecule were selected and their correspondingpeptides were synthesized: SEQ ID No: 1-1864 (FMDIIGVKKV), SEQ ID No:2-1865 (QLDATQLAQV), SEQ ID No: 3-1866 (KLLELTVYNC), SEQ ID No: 4-1868(KMMEDIMNA), SEQ ID No: 5-1869 (AMHDGETQV), SEQ ID No: 6-1871(QLQERLIEL), SEQ ID No: 7-1872 (MLYLTLGSL) and SEQ ID No: 8-1873(KMVEYKSHL). The FIG. 13, tables 1 and 2, includes scores for thedifferent peptides in the mentioned software.

To determine the binding capacity to the HLA-A*0201 molecule, it wasperformed binding tests to T2 cells, having a low expression capacity oftransport-antigen molecules. The results, shown in FIG. 9, indicate thatall the testing peptides performed a good or very good binding affinityto the HLA-A*0201 molecule, being in some cases superior to theHB-ENV₃₃₄₋₂₄₂ peptide affinity used as control, for which a percentageof 100% binding is expected.

The analysis of the effective presentation capacity of these epitopes inthe context of an experimental immunization with the testing molecules,took place in C57BL/6-A2/K^(b) transgenic mice (they were modified toexpress the product of the chimeric gene HLA-A2.1/Kb, where alpha1 andalpha2 domains are the same as the HLA-A*0201 human molecule and thealpha3 domains, both transmembrane and cytoplasmic corresponding to theH-2K^(b) murine molecule) immunized intramuscularly with pPFR1 andpPFR1-HSP70 molecules. Mice injected with saline solution (Sigma) wereused as negative control. Six weeks after the fourth immunization thesplenocytes of the different mice groups were stimulated with thetesting peptides in the context of an ELISPOT assay using ananti-granzyme B antibody as probe. The results obtained (FIG. 10) showthat five of the peptides gave a positive response to granzyme B(activation of antigen-specific CTLs) in vaccinated mice with thechimeric molecule pPFR1-HSP70 and one in those immunized with themolecule containing the isolated PFR1 gene. These results demonstratethat the above testing molecules are efficiently processed and presentedin the context of the MHC-Class I. Furthermore it points that inparticular those epitopes for which positive values are obtained arerecognized by cytotoxic T lymphocytes (CTLs) of the immunized mice.

The analysis of the capacity to generate cytotoxic response in thecourse of an experimental infection with L. infantum was evaluated inC57BL/6-A2/Kb transgenic mice infected via i.v. with 10⁶ infectivepromastigotes of L. infantum (strain JPCM5). 170 Days after infectionmice were sacrificed and their splenocytes or their not-parenchymalliver cells were exposed to the texting peptides in the context of anELISPOT assay using Granzima B as detector antibody. The results show(FIGS. 11 and 12) that four of the tested peptides gave a positiveresponse to Granzyme B in splenocytes and three of them also innot-parenchymal liver cells. Therefore, these peptides are presented tothe CTLs in the course of experimental infection by the parasite.Interestingly, only one out of these four peptides also gave positiveresponse in immunized mice, showing that the antigen presentationdiffers between immunization with plasmids and the course of theexperimental infection.

Concluding Remarks

Example 1 conclusion: the results show that immunized mice with thetesting molecules and subsequently challenged, do not present parasitesin bone marrow, while those groups inoculated with the pCMV4 emptyplasmid or saline control (control) present a high parasite load in thesame tissue after 28 days post-infection. In addition, the parasite loadin spleen and liver of immunized mice are between two to four orders ofmagnitude lower than the detected in the control groups (empty pCMV4plasmid or saline solution). Immunized mice with the pPFR1-H70T moleculeand Leishmania-infected, do not show parasites in spleen after 14 dayspost-infection.

Example 2 2.1 Antigen-Specific Humoral Response.

As an example, it is shown the results obtained after intramuscularlyimmunization of 12-mice-groups of the C57BL16 strain, with 100 μg of thedifferent testing plasmids, as well as the negative controls (miceinoculated with saline solution). In this case, the tested plasmidscontain the sequence corresponding to the fragment of the PFR1 proteinof Leishmania infantum (436 amino acids length) comprised between aminoacids 160 and 595 of the PFR1 protein. Each mouse was immunized 4 timesevery other week.

To analyze the generated humoral response in different groups of mice,blood collection was made two weeks after each immunization and antibodylevel detection in animal sera was measured. The results indicate thatthe presence of anti-PFR1 IgG antibodies appears 15 days post-fourthimmunization in the immunized group with the isolated 436aaPFR1 gen andtwo weeks after the third immunization in those which carry fused436aaPFR1-HSP70 genes. FIG. 14 (A) shows the results for each testingmolecule at 14 and 40 days post-fourth immunization. IgG levels in serafrom immunized mice with the 436aaPFR1 gen fused to the HSP70 gene werehigher than those detected in the group immunized with the isolated436aaPFR1 gene, with a maximum of antibodies within two weekspost-fourth immunization. In all cases, the level of anti-PFR1antibodies slightly descends at six weeks post-fourth immunization.Analysis of isotypes (B) shows a clear polarization of the responsetowards the IgG2a isotype (Th1 type immune response) in releasedantibodies.

2.2 Antigen-Specific Cell Response

Lymphoproliferation tests were carried out three weeks after the fourthimmunization. The spleens were extracted in sterile conditions and theobtained splenocytes were in vitro cultivated in the presence of anincreasing concentration of the rPFR1 protein (0.4, 2 and 10 μg/ml). Inaddition, a mitogen (ConA) and unstimulated splenocytes were included inthis assay as a positive and negative control, respectively. From theresults obtained, shown in FIG. 15, a significant cellular proliferationrate is observed as the recombinant PFR1 (rPFR1) is present and, inaddition, this stimulation is dose-dependent in the splenocytes of miceimmunized by the recombinant testing molecules. Interestingly, thisindex was significantly higher in the groups receiving the PFR1 genefused to the HSP70 and HSP70T. The proliferation rate (IE) for thesplenocytes of mice inoculated with these fusion molecules areapproximately 22, while the measured index for splenocytes from miceimmunized with the plasmid containing the isolated PFR1 gene is about18. In both cases, above the controls; saline solution, IE=4. Theseproliferation rates of splenic cells of mice immunized with the testingmolecules remain with similar values after eight weeks post-fourthimmunization.

The analysis of the effective presentation capacity of these epitopes inthe context of an experimental immunization with the testing molecules,took place in C57BL/6-A2/K^(b) transgenic mice (they were modified toexpress the product of the chimeric gene HLA-A2.1/Kb, where alpha1 andalpha2 domains are the same as the HLA-A*0201 human molecule and thealpha3 domains, both transmembrane and cytoplasmic corresponding to theH-2K^(b) murine molecule) immunized intramuscularly with 436aaPFR1 and436aaPFR1-HSP70 molecules. Mice injected with saline solution (Sigma)were used as negative control. Six weeks after the fourth immunizationthe splenocytes of the different mice groups were stimulated with thetesting peptides in the context of an ELISPOT assay using ananti-granzyme B antibody as probe. The results obtained (FIG. 16) showthat five of the peptides gave a positive response to granzyme B(activation of antigen-specific CTLs) in vaccinated mice with thechimeric molecule 436aaPFR1-HSP70 and one in those immunized with themolecule containing the isolated 436aaPFR1 gene. These resultsdemonstrate that the above testing molecules are efficiently processedand presented in the context of the MHC-Class I. Furthermore it pointsthat in particular those epitopes for which positive values are obtainedare recognized by cytotoxic T lymphocytes (CTLs) of the immunized mice.

Conclusion Example 2

the results show that the 436 amino acids fragment of the PFR1 proteinperforms very similarly to the complete protein, getting a similarpattern of immune response (humoral, cellular and cytotoxic). Therefore,since these parameters are those involved in protection against theparasite, we can conclude that this fragment behaves in all terms tostudy as the complete protein.

1. A nucleotide sequence coding for a PFR1 protein of Leishmania infantum, or a fragment thereof, comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is capable of inducing an antigen-specific T cell cytotoxic immune response in an animal against kinetoplastids causing leishmaniasis disease.
 2. The nucleotide sequence according to claim 1 coding for the 1-595 amino acids of the Leishmania infantum PFR1 protein, determined by SEQ ID NO:
 12. 3. The nucleotide sequence according to claim 1 coding for the 160-595 amino acids of the Leishmania infantum PFR1 protein, determined by SEQ ID NO:
 9. 4. The nucleotide sequence according to claim 1 coding for the 160-548 amino acids of the Leishmania infantum PFR1 protein, determined by SEQ ID NO:
 10. 5. The nucleotide sequence according to claim 1 coding for the 160-385 amino acids of the Leishmania infantum PFR1 protein, determined by SEQ ID NO:
 11. 6. A chimeric molecule comprising: (a) at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal, wherein the at least one nucleotide sequence is fused to (b) a nucleotide sequence coding for a Hsp70 protein of Trypanosoma cruzi, or for a fragments thereof, wherein the chimeric molecule is capable of inducing an antigen-specific T cell cytotoxic immune response in an animal against the kinetoplastids causing the leishmaniasis disease.
 7. The chimeric molecule according to claim 6, wherein the nucleotide sequence coding for the Hsp70 protein of Trypanosoma cruzi, or for the fragment thereof, is determined by SEQ ID NO: 13 or fragments thereof.
 8. A recombinant vector comprising: (a) at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal, or (b) a chimeric molecule, comprising: at least one nucleotide sequence from (a) fused to a nucleotide sequence coding for the HsD70 protein of Trypanosoma cruzi, or for a fragment thereof, wherein the chimeric molecule is capable of inducing an antigen-specific T cell cytotoxic immune response in an animal against the kinetoplastids causing the leishmaniasis disease; or comprising at least one nucleotide sequence from (a) fused to a nucleotide sequence coding for the Hsp70 protein of Trypanosoma cruzi determined by the SEQ ID NO: 13 or a fragment thereof.
 9. The recombinant vector of claim 8, as part of a recombinant plasmid comprising the vector.
 10. A pharmaceutical composition comprising: (a) an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal, or (b) a chimeric molecule, comprising an expression product of at least one nucleotide sequence from (a) fused to an expression product of a nucleotide sequence coding for the Hsp70 protein of Trypanosome cruzi, or for a fragment thereof, wherein the chimeric molecule is capable of inducing an antigen-specific T cell cytotoxic immune response in an animal against the kinetoplastids causing the leishmaniasis disease; or comprising an expression product of at least one nucleotide sequence from (a) fused to a nucleotide sequence coding for the Hsp70 protein of Trypanosoma cruzi determined by the SEQ ID No: 13 or a fragment thereof.
 11. A method for treatment and/or prevention of kinetoplastid infections that cause leishmaniasis disease in an animal, comprising, use of an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum or a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.
 12. The method of claim 11, wherein the animal is a human or a dog.
 13. A method for the manufacture of a therapeutic or preventive vaccine, comprising use of an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.
 14. The method of claim 13, comprising the steps of: (a) identifying at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response in the animal against the kinetoplastids causing the leishmaniasis disease; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in the animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in the animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in the animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against the kinetoplastids causing leishmaniasis disease in the animal, (b) amplifying the at least one nucleotide sequence identified in (a) by PCR using genomic DNA from the kinetoplastid that causes the leishmaniasis disease, and oligonucleotides containing restriction enzymes sites that allow direct cloning of the amplicon into prokaryotic and/or eukaryotic expression vectors after its digestion with these restriction enzymes, (c) cloning that least one nucleotide sequence amplified in (b) for the production of the protein encoded by the at least one nucleotide sequence, (d) purifying the at least one protein(s) obtained in (c) by affinity chromatography, and (e) producing the vaccine for a safe inoculation.
 15. A method for measuring a degree of infection of kinetoplastids that cause leishmaniasis disease in an animal, comprising use of a marker comprising an expression product of a nucleic acid sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum of a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.
 16. The method of claim 15, wherein the animal is a human or a dog.
 17. A method for generating protective immunological memory against infection of kinetoplastids causing leishmaniasis disease in an uninfected animal, comprising use of an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum or a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.
 18. The method of claim 17, wherein the animal is a human or a dog.
 19. A method for clarifying or generating partial or total clearance of kinetoplastids causing leishmaniasis disease of the tissues in an infected animal, comprising use of an expression product of at least one nucleotide sequence selected from the group consisting of: a nucleotide sequence coding for the PFR1 protein of Leishmania infantum or a fragment thereof comprising at least one immunodominant epitope selected from the epitope group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the immunodominant epitope is able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 1-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 12, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-595 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 9, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; a nucleotide sequence coding for the 160-548 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 10, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal; and a nucleotide sequence coding for the 160-385 amino acids of Leishmania infantum PFR1 protein, determined by the SEQ ID NO: 11, and comprising at least one immunodominant epitope able to induce an antigen-specific T cell cytotoxic immune response against kinetoplastids causing leishmaniasis disease in an animal.
 20. The method of claim 19, wherein the animal is a human or a dog. 